The enteric nervous system comprises a complex and widespread network within the gastrointestinal tract and is characterized by both intrinsic and extrinsic arms containing neuron bodies within and outside of the intestine, respectively. Consistent with their role in gut physiology, impaired EAN function can lead to pathologies associated with defective secretory and motor function and chronic inflammatory conditions. EANs cohabitate the intestinal tissue with large populations of immune cells and both, immune and neuronal cells are equipped with sensing mechanisms that monitor perturbations at the luminal surface. Bidirectional interactions between immune and neuronal cells have been documented at steady state and dysfunction in these interactions have been proposed to be part of several disease processes, both local (e.g. irritable bowel syndrome) and systemic (e.g. multiple sclerosis). Despite its relevance for human physiology, the role played by EANs in tissue maintenance and pathology or how EANs communicate luminal insults to the local or distant tissues remains unclear. Novel approaches to gain genetic access to neuronal populations within the CNS have highlighted the transformative potential of these techniques. Surprisingly, little of the progress made in the study of the CNS has been translated into a significant understanding of the peripheral nervous system including EANs. By combining novel imaging and transcriptomic tools, our lab has developed extensive experience in understanding mucosal and intestinal immune responses, and our recent work has highlighted the role that EANs play in orchestrating immune responses. For instance, we uncovered an unexpected role for EANs in modulating a structurally coupled macrophage population via extrinsic sympathetic neuron-derived norepinephrine signaling through adrenergic receptor beta 2 (?2AR) on gut macrophages. To overcome obstacles in the study of EANs mentioned above, this proposal incorporates recent advances in cell-specific actively translating ribosome profiling, tissue clearing, opto- and chemo-genetic modulation of neuronal function as well as viral tracing in order to generate the first functional mapping of EANs, defining microbial sensing circuits in the intestine. Experiments proposed here will not only map this sensing circuit, but also establish tools to manipulate EAN activity to build a functional map of EANs in response to luminal challenges. Since we also propose to employ these novel techniques using human intestinal samples, the three- dimensional comparisons between mouse and human samples will yield insights not only into evolutionarily conserved mechanisms and pathways relevant to EAN architecture and behavior, but also add a strong translational component for understanding human intestinal physiology and pathology. The proposed project will thereby provide a much-needed platform to understand and explore novel therapeutic strategies for the treatment of disorders associated with inflammation-induced neuronal dysfunction.